In recent years clinical diagnosis and detection as well as genetic analysis is being carried out by immunological techniques or hybridization in which specific binding between specific pairs of molecules, such as binding of an antigen to its antibody, is utilized to detect antigens, antibodies, DNA (DeoxyribonucleicAcid) molecules, RNA (RibonucleicAcid) molecules and the like.
Particularly in solid phase binding assay, a method is used that utilizes magnetic particles for detection. A schematic diagram of the conventional solid phase assay using magnetic particles is shown in FIG. 17.
As shown in the figure, the assay is performed using a solid phase 91, molecular receptors 61 that capture an object to be measured 62, a magnetic particle 51, and molecular receptors 63 which detect the object to be measured 62, to thereby assay the object to be measured 62.
The solid phase 91 has a solid phase surface that contacts with a sample solution, and the molecular receptors 61 are immobilized on the solid phase surface. A polystyrene bead, a wall surface of a reaction vessel, a substrate surface or the like is used for the solid phase.
As the molecular receptors 61 and the molecular receptors 63, a molecule that binds specifically to the object to be measured 62 that is present in the sample solution is used. The object to be measured 62 may be an antigen, antibody, DNA molecule, RNA molecule or the like.
The magnetic particle 51 is a labeling material having magnetization. Detecting a magnetic field produced by the magnetization of the magnetic particle makes it possible to determine the amount of the magnetic particle 51 that is in a state to be described hereinafter, and thus the presence or concentration of an object to be measured in the sample solution can be identified. In addition to the magnetic particle 51, a substance that emits a detectable signal, such as a radioactive substance, fluorophore, chemiluminophore, enzyme or the like, may be used as a label. Examples of known assay methods using these labels include Enzyme Immunoassay (EIA) which utilizes the reaction between an antigen and its antibody, or Chemiluminescence (CL) methods such as a strict Chemiluminescence Immunoassay (CLIA) in which a chemiluminescent compound is used as a labeling compound for immunoassay, or Chemiluminescence Enzyme Immunoassay (CLEIA) in which enzyme activity is detected at high sensitivity using a chemiluminescent compound in the detection system.
The molecular receptors 63 that are previously bound to the magnetic particle 51 are antibodies that bind specifically to the object to be measured 62 that is previously bound to the magnetic particle 51.
In the assay illustrated in the figure, first, a sample solution containing the object to be measured 62 is introduced onto the solid phase 91 on which the molecular receptors 61 were immobilized beforehand, whereby the object to be measured 62 specifically binds to the molecular receptor 61. Other substances contained in the sample solution float in the sample solution without binding to the solid phase 91. Then, the magnetic particle 51 on which the molecular receptors 63 are immobilized is introduced into the sample solution. Alternatively, the magnetic particle 51 on which the molecular receptors 63 are immobilized may be introduced into the sample solution at the same time as the object to be measured 62. Thereby, the molecular receptor 63 binds specifically to the object to be measured 62 that is bound specifically to the molecular receptor 61 immobilized on the solid phase. The magnetic particle 51 having the molecular receptors 63 immobilized thereon is referred to as a “magnetic molecule”. Then, a magnetic field produced by the magnetic particle is detected, whereby the amount of magnetic particles 51 bound to the surface of the solid phase 91 is determined. Thus, it is possible to determine the concentration or position of the object to be measured 62 bound to the surface of the solid phase 91. With respect to detection of the magnetic field, detection methods using magnetoresistive elements arranged in an array are disclosed in U.S. Pat. No. 5,981,297 and International Patent Publication WO 97/45740.
In addition to the sandwich assay method described above in which an object to be measured binds specifically to a molecular receptor and a different molecule label then binds specifically to the object to be measured, examples of other assay methods utilizing the above labels include a competitive assay in which an object to be measured and another molecule label competitively bind to a molecular receptor.
In the conventional methods, a signal such as fluorescence or the like from a label is detected by an apparatus, such as an optical detection apparatus, that is capable of detecting the signal. In these methods, it is necessary to capture only the signal from the label of a molecule that is specifically bound to a binding molecule immobilized on a solid phase surface. However, in the case of optical detection, if an unbound labeled molecule is present, the signal from this label may also be captured and thus an accurate assay cannot be conducted.
Accordingly, it is necessary to completely wash away unbound labeling molecules. Further, since it is necessary for an optical detection apparatus to detect a faint light signal, it is difficult to produce a miniturized or low cost detection apparatus.
In the method for detection by magnetoresistive elements using magnetic particles as labels as disclosed in the above U.S. Pat. No. 5,981,297, it is not necessary to wash away any unbound labeling molecules. However, a detection chip on which magnetoresistive elements are arranged in an array requires the switching circuits in order to independently extract the signal of individual elements. Electrical wiring is then required respectively from each of the elements in the array to the switching circuits. Consequently, one problem therewith is that as the number of elements increases the wiring becomes more complicated and the area occupied by the wiring also increases, and thus miniaturization is difficult.
In the aforementioned International Patent Publication WO 97/45740, a circuit for detection of magnetic particles comprises a bridge circuit composed of magnetoresistive elements and transistors functioning as switching elements. However, since magnetoresistive elements require magnetic material, after fabricating a part of the circuit containing transistors by a standard integrated circuit production process, it is then necessary to conduct processes for forming and processing a magnetic thin film.
It is an object of the present invention to provide a biosensor that eliminate the need of washing away of unbound labeling molecules by analyzing an object to be measured such as an antigen, antibody, DNA molecule or RNA molecule through detection of a magnetic field, in which the biosensor is small in size, low in price, and high in sensing accuracy, as well as a method for measuring a magnetic molecule and a method for measuring an object to be measured.